Evolutionary and biochemical analyses reveal conservation of the Brassicaceae telomerase ribonucleoprotein complex

The telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs this role through the addition of repetitive DNA to the ends of chromosomes. The telomerase enzyme is composed of a reverse transcriptase (TERT), which utilizes a template domain in an RNA subunit (TER) to reiteratively add telomeric DNA at the ends of chromosomes. Multiple TERs have been identified in the model plant Arabidopsis thaliana. Here we combine a phylogenetic and biochemical approach to understand how the telomerase RNP has evolved in Brassicaceae, the family that includes A. thaliana. Because of the complex phylogenetic pattern of template domain loss and alteration at the previously characterized A. thaliana TER loci, TER1 and TER2, across the plant family Brassicaceae, we bred double mutants from plants with a template deletion at AtTER1 and T-DNA insertion at AtTER2. These double mutants exhibited no telomere length deficiency, a definitive indication that neither of these loci encode a functional telomerase RNA. Moreover, we determined that the telomerase components TERT, Dyskerin, and the KU heterodimer are under strong purifying selection, consistent with the idea that the TER with which they interact is also conserved. To test this hypothesis further, we analyzed the substrate specificity of telomerase from species across Brassicaceae and determined that telomerase from close relatives bind and extend substrates in a similar manner, supporting the idea that TERs in different species are highly similar to one another and are likely encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able to complement loss of function tert mutants in vivo, indicating TERTs from other species have the ability to recognize the native TER of A. thaliana. Finally, we immunoprecipitated the telomerase complex and identified associated RNAs via RNA-seq. Using our evolutionary data we constrained our analyses to conserved RNAs within Brassicaceae that contained a template domain. These analyses revealed a highly expressed locus whose disruption by a T-DNA resulted in a telomeric phenotype similar to the loss of other telomerase core proteins, indicating that the RNA has an important function in telomere maintenance.


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The telomerase ribonucleoprotein complex (RNP) is essential for genome stability and performs 25 this role through the addition of repetitive DNA to the ends of chromosomes. supporting the idea that TERs in different species are highly similar to one another and are likely 41 encoded from an orthologous locus. Lastly, TERT proteins from across Brassicaceae were able homozygous mutants showed no reduction in telomere length as measured by telomere 150 restriction fragment length analysis (TRF) (Fig 1B). We then propagated both ter1 homozygous 151 template nulls for several generations and measured telomere length in order to determine 152 whether there was progressive loss over time. We observed no reduction in telomere length for 153 either template-null mutant allele, regardless of generation ( Fig 1B) telomere maintenance [28,29]. With the exception of the branches leading to C. rubella (iii) and

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S. irio/B. rapa (vi) for DYSKERIN and A. lyrata KU70, we found no evidence of positive selection 210 (S1 Figure). Thus, contrary to our initial expectations, the protein components of telomerase are 211 not responding at the molecular level to genome duplications or contractions, such as those that 212 occurred in species like B. rapa. These findings support the idea that a highly conserved TER 213 locus may be present and functional in Brassicaceae.

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An evolutionary analysis of Brassicaceae telomerase enzymology recapitulates the 215 accepted organismal phylogeny. We next sought to take an indirect, but more fine-scale shared ancestry for all Brassicaceae) or multiple independent evolutionary events (i.e.,

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alternative TER loci in each species that converged rapidly on a similar TER structure).

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To distinguish between these two possibilities, telomerase substrate utilization was 228 performed in ten species across Brassicaceae ( profile was then compared against the phylogenetic tree reflecting the known relationships 240 among these species (Fig 3). product were determined by comparing against the shortest permutation (N15-GGG). Observed 248 product differences relative to N15-GGG were calculated for all species and oligo combinations.

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The intensity with which the numbered boxes are shaded corresponds to the degree to which 250 the results differ from A. thaliana.

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The substrate utilization profiles we generated for Brassicaceae telomerase closely 252 recapitulated the evolutionary history of the family (Fig 3). The Arabidopsis clade, along with C.
253 rubella, all utilized the suite of oligos in the same manner (White boxes; Fig 3). Importantly, A.  Figure), and telomerase activity in these 279 complementation lines, we did not observe telomere elongation with these constructs (S3 280 Figure). However, with the exception of the 1L chromosome arm in C. rubella, there was no 281 significant decrease in telomere length between the two generations we tested (S3B Figure),

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suggesting some degree of partial complementation.

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These data may represent an inability on the part of some of these

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The resulting reads were mapped to the A. thaliana genome (TAIR 10) and long non-coding 309 RNAs (lncRNAs) were identified using Evolinc (Fig 5A)

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To confirm the involvement of this lncRNA in telomere maintenance, we obtained T-DNA 325 mutants of AtTR/R8 and confirmed homozygosity. We grew these lines for three generations, 326 measuring telomere length using a TRF with each generation (Fig 5C). We found that

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AtTER1/2-like locus partially overlaps RAD52-1A in each of these species, therefore we 340 performed RT-PCR on RAD52-1A in each species in order to map the intron-exon boundaries 341 (Fig 6A). Following cloning and sequencing of RAD52-1A mRNA, we designed reverse primers 342 that bind within the first intron of RAD52-1A and used it in combination with a forward primer in 343 the predicted 5' UTR ( Fig. 6A and 6B). We amplified and sequenced transcripts from RT-PCR products generated using this forward primer with a reverse primer in the first intron of RAD52-

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By selfing these individuals we obtained a population segregating the mutations for both genes 401 allowing direct comparisons between full siblings in TRF analyses.

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Nuclear Protein Isolation, TRAP, TRF and RT-PCR. Nuclear extracts were obtained from 403~10g of seedling tissue as described elsewhere [14]. TRAP was performed as described fragment (TRF) length analyses were performed as described in Nelson et al. (2014). In brief,